The G2 phase of the cell cycle separates S phase (DNA replication) from M phase (chromosome segregation). This cell cycle phase is thought to provide time to the cell to ensure that its genome is properly replicated before it irreversibly commits itself to enter mitosis. Thus, replication errors and DNA damage will delay cells in G2 until the defects are properly repaired. Recent evidence suggests that other events such as the integrity of the microtubule cytoskeleton is also monitored during G2 by the chfr checkpoint. During this funding period, our PO1 project has revealed that the assembly of the kinetochore is an event that also occurs in G2. Although kinetochores function specifically during mitosis to ensure chromosomes are properly attached to spindle microtubules, this macromolecular complex is assembled and disassembled every mitosis. Examination of a large number of kinetochore proteins revealed that their assembly onto kinetochores follow a discrete temporal pattern that occurs during G2. Thus, the time of appearance of specific proteins to kinetochores may define discrete points in G2. We discovered that disruption of the assembly pathway delays cells in G2 for a significant length of time. In collaboration with the project by Muschel, we found that cells with DNA damage arrested uniformly at a discrete point in G2 that is defined by the CENP-F kinetochore protein. The combined data suggest that kinetochore assembly is a critical event during G2 that appears to be monitored by a checkpoint. Lastly, we have established a link between defects in chromosome alignment and cell death. In the absence of hNuf2 kinetochore protein, chromosomes fail to align yet they die instead of arresting in mitosis. We discovered that the unusually rapid cell death may be due to the absence of survivin at kinetochores that are depleted of hNuf2. The broad goal of this renewal application is to understand the molecular details about how kinetochores are assembled in G2 and how kinetochore defects can lead to cell death. Our hope is that these studies will provide novel aspects about the G2 phase of the cell cycle that may have applications for developing methods for enhanced cell killing by DNA damaging agents.